Today, lung cancer is the leading cause of death in both men and women in industrialized countries, accounting for an estimated 28% of all cancer deaths in the United States. Non-small cell lung cancers (NSCLC) represent the majority of lung cancers and carry a poor prognosis with a median survival of less than 12 months. Most patients present with unresectable disease and current treatment options of chemotherapy and radiation are palliative at best. Therefore, new strategies are needed in the treatment of NSCLC in order to impact this disease. In this study, we are focusing on NSCLC models for examining distal signaling mechanisms that modulate the chemotherapy sensitivity, generation, and maintenance of NSCLC cells/tumors. Specifically, the grant application focuses on the cell signaling pathways regulating both hnRNP L function and the alternative splicing of caspase 9. The expression of caspase 9 is regulated by alternative splicing via the inclusion or exclusion of a four exon cassette (exons 3, 4, 5, and 6). Inclusion of this exon cassette into the mature transcript produces the pro-apoptotic caspase 9 (caspase 9a) while the exclusion produces the anti-apoptotic caspase 9b. Studies from our laboratory have demonstrated that NSCLC tumors present with a dysregulated ratio of caspase 9/caspase 9b analogous to an anti-apoptotic/chemotherapy resistance phenotype. Subsequent studies by our laboratory demonstrated that the alternative splicing of caspase 9 had important functions in anchorage-independent growth (AIG) in NSCLC cells, AIG induced by EGFR mutation in non-transformed human bronchial epithelial cells, and erlotinib sensitivity. Mechanistically, our laboratory identified an exonic splicing silencer (C9/E3-ESS) in exon 3 that regulates the inclusion of the exon 3, 4, 5, and 6 cassette of caspase 9 pre-mRNA. hnRNP L was shown to associate with this RNA cis-element, and repress the inclusion of the exon cassette. Importantly, phosphorylation of hnRNP L on ser52 (observed only in transformed cells) was required for repression of the exon 3,4,5,6 cassette. Lastly, ser52 phosphorylation of hnRNP L was shown as a required mediator of the tumorigenic capacity of NSCLC cells via the alternative splicing of caspase 9. These key mechanisms are specific to transformed cells, translatable to >70% of NSCLCs, and at an extreme distal point in oncogenic pathways. Therefore, these distal mechanisms are plausible and highly desired targets for the development of new anti-cancer therapeutics. The overall goal of this study is to determine the mechanisms and cell signaling pathways regulating the definition of exon 3, and thus the alternative splicing of caspase 9. Furthermore, we are proposing pre-clinical studies to determine whether these mechanisms as well as specific targeting of caspase 9b are effective targets for treating NSCLC as well as enhancing the effectiveness of current chemotherapeutic agents used in the clinic.